Basic introduction of Real-time PCR and
LightCycler^® 480 system

Jing-yuan Chen

Graduate Institute of Veterinary Pathobiology

08 16, 2019

Online slide

檔案網址
https://lazyliondog.github.io/realtimePCR/
簡要中文操作手冊按此下載

Polymerase chain reaction
(PCR)

PCR

Thermal cycle

Source: Polymerase chain reaction

Amplification curve

Four pahses for PCR

Ground
Expoential
Linear
Plateau

2ⁿ / cycle

PCR stage

Reference DOI: 10.1100/tsw.2011.124.

Crucial factors for PCR efficiency

Quality of DNA template
DNA polymerase reality
dNTP concentration
Primers design, concentration…
Many other factors!
- Theoretically!

Real-time PCR

Real-time PCR machines

Roche	Bio-Rad	Applied Biosystems	Qiagen
LightCycler^®480	CFX96 Touch^TM	StepOne^TM	Rotor-Gene Q

Basic introduction

Applications

Gene detection	Gene expression
Species identification Pathogens screening Diseases diagnosis Antibiotic resistance gene	Genotyping Food safety analysis Gene knockdown Pharmacy researches

Trends of publications using Real-time PCR

qPCR Trend

Number of publications in PubMed related to qPCR.
(Number of publications in PubMed related to specific qPCR applications.)

Reference DOI: 10.1016/j.bdq.2014.08.002

qPCR for food analysis

Summary of the research articles published in different journals.
The data was retrieved from Scopus by using the key word “Real-Time PCR for Food Analysis”

Reference DOI: 10.1007/s13197-016-2205-0

Two major detection format

Non-specific dsDNA binding dyes
Sequence-specific fluorescence detection chemistry

SYBR green I

SYBR green 1

Indiscriminate binding into any dsDNA

Primer dimers

Sample DNA quality

Melting curve analysis (Tm calling analysis)

Reference DOI: 10.1038/sj.leu.2402922

Melting curve analysis

Reference DOI: 10.1002/9780470027318.a1406.pub2

Hybridization probe

Fluorescence Resonance Energy Transfer (FRET)

FRET

TaqMan probe

TaqManProbe

Reference DOI: 10.1038/sj.leu.2402922

$all format$

Reference DOI: 10.1007/s13197-016-2205-0

How to design my real-time PCR?

Resource

Free websites

	線上 primer 設計 http://bioinfo.ut.ee/primer3-0.4.0/
$Beacon$	線上 primer 設計 http://www.premierbiosoft.com/qOligo/Oligo.jsp?PID=1
	檢查目標產物結構 http://unafold.rna.albany.edu/?q=mfold/download-mfold
	多重序列比對 https://www.ebi.ac.uk/Tools/msa/clustalo/ Reference DOI: 10.1002/pro.3290

Example (I)

曾經有過這樣的設計 (142-232)，看起來好像不錯！？

CSFV qPCR

Exapmle (II)

結果，因為立體結構的關係，使得擴增效率非常差！

設計實驗以後，利用免費資源看一下可能的狀況！

Structure1

Data analysis

Reference DOI: 10.1016/j.bdq.2014.08.002

Quantification

Cp calculation
Abosolute
Relative
Tm calling analysis (Melting curve analysis)

Cp calculation

Crossing poing (Cp) = Cycle quantification (Cq) = Cycle threshold (Ct) = Take-off point (TOP)
各家儀器設備設定值不同而有所差異！！
MIQE

qPCR_Ct

Picture source:What Is A Cycle Threshold (Ct) Value In qPCR?

PCR efficiency

Reference: http://www.gene-quantification.de/pfaffl-qPCR-efficiency1.gif

PCR efficiency (I)

PCR product number at the number of Cp

$N = N_{0}\times E^{Cp}$

Linearised

$LogN = LogN_{0}+Cp\times LogE$

Calculate the “Cp” and describe the PCR amplification

$Cp=-(\frac{1}{LogE})LogN_{0}+\frac{LogN}{LogE}$

Slope of this equation!
Draw the plot…

PCR efficiency (II)

PCR efficiecy “E” can be described as

$E = 10^{-\frac{1}{Slope}}$
$E(\%) = -1 + 10^{-\frac{1}{Slope}}$

Absolute quantification

Second-derivative maximum method: Standard method
Fit point method: Optional, user define

Reference DOI: 10.1002/9780470027318.a1406.pub2

Relative quantification (I)

Determination of mRNA expression level
Gene dosage quantification
Relative to reference gene and/or calibrator sample

SA: stalbe angina
ACS: acute coronary syndrome

Reference DOI: 10.5114/aoms.2014.44871

Relative quantification (II)

$Relative\ ratio = \frac{Target\ gene\ concentration}{Reference\ gene\ concentration}$

Reference gene:

Unaffected by experiment conditions (non-regulated)
Wide variety of cellular processes
One or more reference can be used

Reference gene selection

Reference DOI: 10.1016/j.bbrc.2003.11.177 (被引用1,367次)

Relative quantification (III)

Optional:

$Calibrator\ Normalize\ Ratio = \frac{\frac{Target\ gene\ concentration}{Reference\ gene\ concentration}(Sample)}{{\frac{Target\ gene\ concentration}{Reference\ gene\ concentration}(Calibrator)}}$

Calibrator:

Correct the differences in detection sensitivity between Target / Reference gene and several experiments (e.g., long-term studies)
Not treated cell line
Sample before experiment

Relative quantification (IV)

Efficacy = 2 → ($2^{\Delta\Delta CT} method$)
Efficacy adjust (linear) - using standard curve

Relative quantification (V)

Relative quantification

Reference: Roche 教育訓練專員講義

Tm Calling analysis

Tm Calling

Analysis tools

LightCycler 480 Software

LC480 software

Resource fo analysis tools

$Analysis platforms$

The MIQE guidelines
for Real-time PCR

Different results?

Different Melting cruve

Reference DOI: 10.1016/j.mcp.2010.04.002

MIQE

Minimum Information for Publication of Quantitative Real-Time PCR Experiments, (2009)
MIQE & PCR (2016, Jun)

$MIQE 2016$

Ohter guides

$Other guides$

LightCycler^® 480 Instrument

General of LC480

LC480 General

LC480 front
LC480_fans

Enviroment parameters

機器使用時，請將冷氣開啟，以維持溫度 (大約 26˚C 左右)。
冷氣開啟時，請注意隨手關門，電費不便宜！

LightCycler^® 480 (I)

LC480 外觀

LC480 light

LightCycler^® 480 (II)

plate loader 1

plate loader 2

Sample capicity

Whit plate

限定使用Roche原廠耗材！
(盤子與封模，唯一限定的材料)

LightCycler^® 480 (III)

Thermal cycler

Thermal cycler

Thermal base

LightCycler^® 480 (IV)

Optical system

LC480 filters

LightCycler^® 480 - Filter set

Filter set

LightCycler^® 480 - Xenon lamp (氙)

Xenon lamp

一定要遵守這些事

請注意！

使用前登記，安排實驗次序
注意空調，維持機器運作時的室內溫度
限定使用原廠白盤及封膜！ (未遵守者禁止使用)
燈泡很貴！請算好操作的時間，不要開著機器讓他燒！

Basic introduction of Real-time PCR andLightCycler® 480 system